Method Of Inducing Or Modulating Immune Response

ABSTRACT

The use of axotrophin, also known as MARCH VII to induce or regulate immune response to an antigen whether foreign or self, suitably in a vertebrate, for example a mammal is disclosed. Isolated axotrophin and nucleotides and polypeptides encoded by or derived from axotrophin, compositions containing one or more thereof and assay methods are provided as further aspects of the invention.

The present invention relates to the use of a polynucleotide,polypeptide and proteins encoded by or derived from such polynucleotide,along with uses for the polynucleotide, polypeptide and proteins and toa method of inducing or modulating immune response to an antigen andfurther relates to a method of determining the immune status of anindividual with respect to a given antigen. In particular, the inventionrelates to the use of axotrophin, also known as MARCH VII to induce orregulate immune response to an antigen whether foreign or self, suitablyin a vertebrate, for example a mammal. The invention also providesisolated axotrophin and nucleotides and polypeptides encoded by orderived from axotrophin, compositions containing one or more thereof andassay methods.

As used herein, reference to axotrophin includes a reference to apolynucleotide or polypeptide sequence having at least 75% andpreferably at least 90% sequence identity to an identifying sequence ofaxotrophin. The finding that axotrophin plays a significant role in theimmune response of an individual enables its use in numerousapplications in a variety of techniques known to those skilled in theart of molecular biology, such as use as hybridization probes, use asprimers for PCR, use in an array, use in computer-readable media, use insequencing full-length genes, use in the recombinant production ofprotein, and use in the generation of anti-sense DNA or RNA, theirchemical analogs and the like.

Identified polynucleotide and polypeptide sequences have numerousapplications in, for example, diagnostics, forensics, gene mapping,identification of mutations responsible for genetic disorders or othertraits, to assess biodiversity, use as primers in expression assays andto produce many other types of data and products dependent on DNA andamino acid sequences. Axotrophin is known and details of the axotrophingene may be found in the GenBank database and elsewhere under variousAccession Numbers including AK022973 and NM_(—)022826.2 (human) andAF155739 and NM_(—)020575 (murine). Human axotrophin protein sequencemay be found under Accession Number NP_(—)073737.1 and murine axotrophinprotein under NP_(—)065600.1. These sequences are set out below asSequence Idenitification Numbers 001 to 004 respectively. Axotrophin isone of 216 genes identified as being enriched in mouse embryonic, neuraland hematopoietic stem cells as disclosed in Science, Vol 298, 597-60018 Oct. 2002 and is said (in Table 1) to participate in signaling andthe ubiquitin pathway. Genes & Development 15:2660-2674 published in2001 discloses that mouse protein axotrophin has a RING-CH domain and isrequired for normal brain development and that disruption of theaxotrophin gene may result in neural degeneration and callosal agenesis.There would appear to be little else known about the function ofaxotrophin from the published literature.

The present inventor has now found that axotrophin induces or regulatesimmune response to an antigen at the genomic, mRNA and/or protein level.It is believed regulation may be manipulated through antisense DNA orRNA or binding molecules. Additionally, axotrophin has been found toregulate T lymphocyte cell proliferation and to regulate release ofleukemia inhibitory factor (LIF) for example from activated T lymphocytecells as set out in the Examples below. WO 03/052424 discloses thatc-kit (CD117), STAT3, stem cell factor (SCF) and LIF are elevated intolerant immune responses and that these may be used in modulatingimmune response generated to an antigen. A LIF murine sequence isavailable at SWISSPROT P09056. A human sequence is available atSWISSPROT P15018.

The invention provides the use of axotrophin or a polypeptide orpolynucleotide encoded by or derived from axotrophin to induce or toregulate directly or indirectly the immune response to an antigen,whether a “foreign” antigen (for example allogeneic, xenogeneic,procaryotic, viral or synthetic) or autologous (“self”) antigen.

Manipulation of the immune response may be in ex vivo, in vivo or invitro cell population.

Any reference to “regulation” of the immune response in relation to thisinvention includes regulating phenotypic development and maintenance ofcell populations that regulate immunity to a given antigen.

Reference herein to materials “derived from” axotrophin includes, by wayof example, anti-sense sequences including RNAi, whether single ormultiple stranded, and small molecules binding to polypeptides orpolynucleotides of axotrophin, including antibody especially monoclonalantibody. Reference to materials derived “directly or indirectly” fromaxotrophin includes any such polynucleotides or small molecules.

Reference herein to “polypeptide” includes protein and especially matureprotein.

The invention also provides the use of axotrophin or a polypeptide orpolynucleotide encoded by or derived from axotrophin in the manufactureof a medicament to induce or to regulate directly or indirectly theimmune response of a vertebrate to an antigen, whether a “foreign”antigen (for example allogeneic, xenogeneic, prokaryotic, viral orsynthetic) or autologous (“self”) antigen.

The medicament produced according to the invention is suitable fortreating an individual to reduce rejection of transplanted tissue, cellsor organ.

The invention further provides for use of axotrophin or a polynucleotideencoded by or derived from axotrophin to regulate expression of LIF. LIFmay induce or regulate directly or indirectly the immune response of avertebrate to an antigen, whether a “foreign” antigen (for exampleallogeneic, xenogeneic, procaryotic, viral or synthetic) or autologous(“sew”) antigen.

Suitably, use of polypeptide or polynucleotide encoded by or derivedfrom axotrophin allows cancerous immune cells that are sensitive to LIFto be targeted ex vivo or in vivo,

Without wishing to be bound by any theory, it is believed thataxotrophin also regulates the expression of Foxp3 and SOCS3 at thegenomic and/or protein level and that this plays a role in T cellregulation.

The invention provides in a further embodiment for use of axotrophin ora polypeptide or polynucleotide encoded by or derived from axotrophin toinduce or regulate T cell proliferation in a cell population in an invivo, ex vivo or in vitro environment The T cells are preferably Tlymphocyte cells.

Advantageously, the present invention may be used to guide the immuneresponse of a vertebrate for example a mammal to accept a transplantedorgan, tissue, cell, gene or gene product, artificial substance, or anyother agent utilized within the body, for example for a therapeuticpurpose. The invention is especially applicable in the use of stem cellsIn therapy or otherwise.

The immune suppressive activity of axotrophin may be used to protectintroduced biological materials from immune attack, for example intransplantation of cells, to treat diseases including neurodegenerativediseases, tissues for grafting or example bone marrow, skin, cartilage,bone, tendons, muscle including cardiac muscle, blood vessels, cornea,neural cells, gastrointestinal cells and others and organs fortransplantation including kidney, liver, pancreas including the Isletcells, heart and lung.

Suitably, expression of the encoded or derived from axotrophinpolypeptide or regulatory polypeptide or polynucleotide sequences thatinfluence axotrophin activity may be modified in the host immune cellsex vivo to bias the immune response to accept the introduced biologicalmaterials, Alternatively, or additionally, expression of axotrophinwithin the biological materials may be modulated ex vivo to carryimmunomodulatory properties when introduced in vivo.

Axotrophin may be employed in the treatment of immune disordersincluding severe combined immunodeficiency (SCID) by regulating, up ordown, T lymphocytes as well as effecting the cytolytic activity of NKcells and other cell populations. These immune deficiencies may begenetic or be caused by viral (for example, HIV) as well as bacterial orfungal infections, or may result from autoimmune disorders, Morespecifically, infectious diseases caused by viral, bacterial, fungal orother infection may be treatable using a protein orpolynucleotideencoded by or derived from axotrophin including infectionsby HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmaniaspp., malaria spp. and various fungal infections such as candidiasis aswell as where a boost to the immune system generally may be desirable,for example in the treatment of cancer.

Autoimmune disorders which may be treated using a protein orpolynucleotide encoded by or derived from axotrophin include, forexample, connective tissue disease, multiple sclerosis, systemic lupuserythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependentdiabetes mellitis, myasthenia gravis, graft-versus-host disease andautoimmune inflammatory eye disease. Such a protein (or antagoniststhereof, including antibodies) of the present invention may also to beuseful in the treatment of allergic reactions and conditions (forexample, anaphylaxis, serum sickness, drug reactions, food allergies,insect venom allergies, mastocytosis, allergic rhinitis,hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopicdermatitis, allergic contact dermatitis, erythema multiforme,Stevens-Johnson syndrome, allergic conjunctivitis, atopickeratoconjunctivitis, venereal keratoconjunctivitis, giant papillaryconjunctivitis and contact allergies), such as asthma (particularlyallergic asthma) or other respiratory problems.

In using axotrophin, down regulation may be in the form of inhibiting orblocking an immune response already in progress or may involvepreventing the induction of an immune response.

The use of axotrophin in down regulating or preventing one or morefunctions during the immune response for example in reducing interferongamma release, may be useful in situations of tissue, skin and organtransplantation and in graft-versus-host disease (GVHD). Up regulatingaggressive immune responses by down modulation of axotrophin or apolynucleotide or polypeptide encoded by or derived from axotrophin isalso useful. Upregulation of immune responses may be in the form ofenhancing an existing immune response or eliciting an initial immuneresponse. For example, enhancing an immune response may be useful incases of viral infection, including systemic viral diseases such asinfluenza and the common cold. Regulation of axotrophin suitablyfacilitates a T cell-mediated immune response against tumour cells.

A polypeptide of axotrophin may be involved in regulating in chemotacticor chemokinetic activity for mammalian cells, including, for example,monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils,epithelial and/or endothelial cells. The invention provides chemotacticor chemokinetic compositions for example proteins, antibodies, bindingpartners, or modulators containing axotrophin or polynucleotides orpolypeptides encoded by or derived from axotrophin, provide particularadvantages in treatment of wounds and other trauma to tissues, as wellas in treatment of localized infections. For example, attraction oflymphocytes, monocytes or neutrophils to tumors or sites of infectionmay result in improved immune responses against the tumor or infectingagent.

The invention may also suitably be used to guide the immune system toallow for acceptance of, or at least reduced aggressive response to, anantigen associated with an autoimmune disease or disorder, whethereliciting the innate or adaptive immune response during the auto-immunereaction.

Further, the invention may be used to guide the immune response toreject an organ, tissue, cell, pathogen such as a prokaryote, yeast orfungus, parasite or virus, a gene or gene product, an artificialsubstance, or any other agent that may invade or be taken into the body,or be generated within the body, wherein that agent is unwanted,diseased (for example neoplastic tissue or infected tissue), orotherwise deleterious to the host patient.

The invention also may be used to enhance the degree of immune responseagainst antigen following vaccination, especially in cases where currentvaccination procedures are off limited success in generating aprotective immune rejection response against biological agents,including for example those associated with germ warfare

An especially advantageous aspect of the invention is the specificity ofresponse generated on activation by a specific antigen. The immuneresponse may be guided to tolerance or aggression by signal pathwaymodulation in vivo. On challenge with an antigen, responsive cells maybe guided towards tolerance or aggression in accordance with variousaspects of the invention and non-responsive cells remain unaffected bythe regulatory adaptation. The target antigen itself triggers responsivecells or responsive cell populations: cells capable of responding onlyto other antigens are not triggered, and are therefore not receptive toguiding towards tolerance or aggression towards the relevant antigen atthat time. As an alternative or supplement, Immune cells may be guidedto regulatory tolerance, or aggression ex vivo. Immune cells, forexample of blood and/or spleen, may be removed, treated with antigen andguided to tolerance or aggression, before being returned to theindividual.

As used herein, the term “antigen” has the meaning generally understoodin the ar and includes any naturally occurring, recombinant or syntheticproduct such as a polypeptide, which may be glycosylated. The termantigen also includes complexes of protein carriers and non-proteinmolecules such as steroids, carbohydrates or polynucleotides.

Antigen is also used herein to refer to any substance which comprises aplurality of antigens and opitopes, for example a cell or tissue, organ,implant, indeed any substance to which an immune response can be mountedby the immune system of a vertebrate, for example a mammal.

The antigen may be an antigen of a pathogenic organism associated withhuman or animal disease. Organisms which cause animal disease includefor example foot and mouth disease virus, Newcastle disease virus,rabies virus and Salmonella species. Organisms which cause human diseaseinclude for example bacteria such as Salmonella species including S.typhimurium and S. typhi, Staphylococcus such as S. aureus, Pertussis,Vibrio cholera, pathogenic E. coli, Mycobacteria species such as M.tuberculosis and M.paratuberculosis. Viral organisms include for exampleHIV-1 or HIV-2 (which include the viral antigens gpl60/120), HBV (whichincludes surface or core antigens), HAV, HCV, HPV (for example HPV-16),HSV-1 or-2, Epstein Barr virus (EBS), neurotropic virus, adenovirus,cytomegalovirus, polio myelitis virus, and measles virus.

Small pox and anthrax are also pathogens of interest and which may besubject to the present invention. Eukaryotic pathogens include yeast,such as C. albicans, aspergillus, schistosomes, protozoans, amoeba,plasmodia, including for malaria, toxoplasma, giardia and leishmania.

The antigen may also be a tumour associated antigen. Such antigensinclude GEA, alpha fetal protein (AFP), neu/HER2, polymorphic endotheliamucin (PEM), N-CAM and Lewis Y.

The antigen may be an abnormally expressed antigen, such as p53 orvirally-modified antigen.

Antigens such as those mentioned above may be obtained in the form ofproteins purified from cultures of the organism, or more preferably byrecombinant production of the desired antigen. Antigens may also beproduced by chemical synthesis, for example employing an automatedpeptide synthesiser such as are commercially available.

Instead of wild-type polypeptide, an appropriate fragment may be usedprovided the desired activity is retained. The skilled person is readilyable to make changes to amino acid sequence of any polypeptide in aconservative manner, for example without abolishing function.

A further aspect of the present invention provides a method ofmodulating an immune response to an antigen in an individual, the methodincluding provision in the individual of axotrophin or a polypeptide orpolynucleotide encoded by or derived from axotrophin.

Such provision may be by administration of the polypeptide orpolypeptides, or may be by administration of polynucleotide encoding thepolypeptide or polypeptides. A further approach comprises administrationof a substance that upregulates expression of the polypeptide orpolypeptides, for example by binding the promoter or other regulatoryelement of the relevant gene.

The present invention also provides for a method of modulating an immuneresponse of an individual to an antigen, the method comprisingadministering a substance that affects activity of axotrophin in theindividual,

The amount of polypeptides expressed directly or indirectly byaxotrophin in the individual may be modulated either upwards, so thatactivity is increased or augmented, or downwards, so that activity isdecreased or reduced. Increased activity is associated with a promotionof immune tolerance, while decreased activity is associated with apromotion of immune response against the antigen, that is an aggressiveresponse.

Thus, in accordance with the present invention there is provided amethod of manipulating the response of the immune system to a givenantigen, for example increasing tolerance of the immune system of anindividual to an antigen, the method comprising administering to theindividual axotrophin or a polypeptide or polynucleotide encoded by orderived from axotrophin or a substance that enhances the amount oractivity of polypeptide expressed directly or indirectly by axotrophin.

Further, in accordance with the present invention there is provided amethod of potentiating or increasing the aggressive response of theimmune system of an individual against an antigen, the method comprisingadministering to the individual a substance that decreases the amount oractivity of a polypeptide expressed directly or indirectly byaxotrophin.

A substance may decrease the amount or activity of polypeptide expresseddirectly or indirectly by axotrophin by binding or otherwise interactingwith it. Such a substance may be for example an antibody molecule withappropriate binding specificity, or other peptidyl or non-peptidylmolecule that binds the polypeptide. Production of the polypeptide, maybe reduced by for example down-regulating promoter function of therelevant gene or by targeting encoding mRNA to reduce translation (forexample by antisense or dsRNA inhibition, RNAi, or ribozyme digestion)or by means of a substance that promotes degradation of the polypeptide,for example using ubiquitination.

A substance may increase activity of polypeptide expressed directly orindirectly by axotrophin by means of binding, for instance by binding toa promoter or enhancer region of an encoding polynucleotide sequence toincrease promoter function.

A further aspect of the invention provides a method of enhancing anaggressive immune response against an antigen in an individual, or ofproviding an enhanced aggressive immune response or reduced aggressiveimmune response, or of promoting tolerance in an individual, the methodcomprising administering to the individual a composition comprising theantigen or polynucleotide encoding the antigen and administering acomposition which comprises a polypeptide expressed directly orindirectly by axotrophin or a substance that alters the amount oractivity of such a polypeptide in an individual.

Two or more compositions may be provided as a combined preparation forsimultaneous or sequential administration.

The level of materials produced on expression of axotrophin, for exampleLIF may be altered, for example via encoding polynucleotide, or byalteration of endogenous expression levels, or by alteration ofpolypeptide activity, for example by means of a small molecule or otheractive agent, so as to modulate the presence or degree of tolerance oraggression that the immune system of an individual shows to an antigenof interest. The present invention may be used in a variety of contexts,including conditioning of the immune system with respect to a plannedtransplant, to potential challenge with a pathogen or other foreignbody, to transformed cells of the host, for example cancer cells orvirally-infected cells, and in an autoimmune disorder.

An aggressive immune response modulated or affected in accordance withthe present invention may be an inappropriate immune response, forexample in an autoimmune disease, or an appropriate immune response, forexample in response to a pathogen.

Axotrophin has been found to provide regulation of the immune responsein a vertebrate for example a mammal including human. Suitably theresponse is a tolerogenic immune response to an antigen in thevertebrate.

In a further embodiment, the present invention provides for use ofaxotrophin or a polypeptide or polynucleotide encoded by or derived fromaxotrophin for assaying immune status. Axotrophin or a polypeptide orpolynucleotide encoded by or derived from axotrophin is suitable foruseful in clinical medicine or veterinary medicine.

The invention also provides a method for determining immune status of anindividual, the method comprising determining the level of expression ofaxotrophin or a polypeptide or polynucleotide encoded by or derived fromaxotrophin in a test sample comprising tissue, cells and/or bodily fluidremoved or obtained from the individual and comparing the level for thetest sample with that of a control sample, wherein a level in the testsample greater than that of the control sample is indicative that theimmune status in the individual comprises a tolerant immune response, orwherein a level in the test sample lower than that of the control sampleis indicative that the immune status in the individual comprises anaggressive immune response.

An assay of immune status may be used to assess immune status of anindividual in relation to immune response to a pathogen, immune responseto a diseased tissue such as a tumour, tolerance to a transplantedtissue, cell or other material (for example to indicate a status oftolerance to an organ allograft or xenograft when it is desired toreduce or remove immunosuppressive therapy to the recipient). Thus, suchan assay may be used in a diagnostic context, to determine the status ofthe immune system of an individual. It may be used to assess the benefitor success of ongoing treatment.

The method is particularly beneficial for determining immune status ofan individual having a tissue or cell transplant and optionally isundergoing therapy Suitably, the level is determined for a test samplecomprising peripheral blood. Reference herein to an “individual”includes animal as well as human.

A further aspect provides for use of axotrophin or a polypeptide orpolynucleotide encoded by or derived from axotrophin or a substance thatalters amount or activity thereof in an individual as disclosed, in themanufacture of a medicament to boost or reduce an aggressive immuneresponse in an individual against an antigen or to alter tolerance ofthe immune system to an antigen, or for use in any method of treatmentas set out herein. Such a medicament is generally for administration fortreatment or prevention of a disease or disorder associated with theantigen, whether the antigen be of a pathogen, disease cell such as atumour, or a material to be transplanted, such as an organ, tissue orcell.

Generally, such a substance according to the present Invention isprovided in an isolated and/or purified form, that is substantiallypure. In a preferred embodiment, the substance is in a composition whereit suitably represents at least 80% active ingredient, preferably atleast 90%, more preferably at least 95% and especially at least 98% byweight of the composition.

A polypeptide encoded by or derived from axotrophin or a peptidylsubstance that affects the activity or amount such a polypeptide, forexample by binding with it (such as an antibody molecule) or by bindingwith a promoter element that affects the polypeptide production byexpression from the encoding gene, or other polypeptide that may be usedin any aspect or embodiment of the present invention, may be produced byrecombinant expression.

A substance to be given to an individual in accordance with anembodiment of the present invention may be administered in a“prophylactically effective amount” or a “therapeutically effectiveamount” as desired. A prophylactic effect may be sufficient topotentiate or reduce an aggressive immune response of an individual to asubsequent challenge with antigen (depending on whether an aggressiveimmune response against antigen of a tolerigenic response is desired).Most preferably the effect is sufficient to prevent the individual fromsuffering one or more clinical symptoms as a result of subsequentchallenge with antigen. A therapeutic effect is sufficient to potentiateor reduce an aggressive immune response of an individual to pre-existingreaction, preferably sufficient to antagonise the reaction, wholly orpartially, for example in an autoimmune disorder or in transplantrejection. Most preferably the effect is sufficient to ameliorate one ormore clinical symptoms. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, for example decisionson dosage etc, is within the responsibility of general practitioners andother medical doctors, and typically takes account of the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to practitioners,Examples of the techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

The invention also provides a ribozyme having specificity for apolynucleotide of the invention based upon the nucleotide sequence ofaxotrophin.

In addition, the invention encompasses methods for the manufacture of amedicament for treating conditions of or related to the immune systemcomprising administering a compound or other substance that modulatesthe overall activity of axotrophin or a polypeptide or polynucleotideencoded by or derived from axotrophin. Compounds and other substancescan effect such modulation either on the level of target gene/proteinexpression or target protein activity.

The invention in a further aspect provides isolated axotrophin apolynucleotide or a polypeptide encoded by or derived from axotrophin,including recombinant DNA molecules, cloned genes or degenerate variantsthereof, especially naturally occurring variants such as splicevariants, allelic variants, antisense polynucleotide molecules, andantibodies that specifically recognise one or more epitopes present onsuch polypeptides, as well as hybridomas producing such antibodies.

The polynucleotide sequences of the present invention also include asegment of axotrophin that uniquely identifies or represents thesequence information of axotrophin. Isolated polynucleotide sequencesmay be produced by cloning the appropriate polynucleotide sequence andexpressing it in a vector according to methods known in the art.

The polynucleotides of the present invention also include apolynucleotide that hybridizes under stringent hybridization conditionsto (a) the complement of axotrophin; (b) a polynucleotide nucleotidesequence encoding axotrophin; (c) a polynucleotide which is an allelicvariant of axotrophin; (d) a polynucleotide which encodes a specieshomolog (for example orthologs) encoded by or derived from axotrophin or(e) a polynucleotide that encodes a polypeptide comprising a specificdomain or truncation of any of the polypeptides encoded by or derivedfrom axotrophin.

As a means of providing the immune response, delivery of a functionalgene encoding polypeptides encoded by or derived from axotrophin toappropriate cells is suitably effected ex vivo, in situ, or in vivosuitably by the use of vectors, and more particularly viral vectors (forexample, adenovirus, adeno-associated virus, or a retrovirus), or exvivo by use of physical DNA transfer methods (for example, liposomes orchemical treatments). Naked DNA or RNA may be used for expression of anencoded gene product in vivo. Naked DNA may be delivered using directinjection or by use of gene-guns (Yang etal., 1990) or any othersuitable technique, such as topically for example for treatment ofpsoriasis. Cells transformed or transfected or otherwise geneticallyengineered to contain axotrophin or a polynucleotide encoded or derivedfrom thereby or to express axotrophin polypeptide may be employed todeliver the functional material.

In a further aspect, the invention provides a vector for the expressionof axotrophin, a polynucleotide sequence or a polypeptide encoded by orderived from axotrophin, the vector containing axotrophin or apolynucleotide sequence encoding axotrophin, for example apolynucleotide sequence complementary thereto or the reverse thereof apromoter sequence and a termination sequence.

Viral vectors may be used to deliver axotrophin or a polynucleotideencoded by it for production, suitably in vivo. Axotrophin or apolynucleotide encoded by axotrophin which encodes a polypeptide orother peptidyl molecule for use according to the present invention maybe used in a method of gene therapy. This requires use of suitableregulatory elements for expression and a suitable vector for deliver ofthe expression unit (coding sequence and regulator elements) to hostcells in vivo. A variety of vectors, both viral vectors and plasmidvectors, are known in the art, see for example U.S. Pat. No. 5,252,479and WO 93/07282 and countess other publications. In particular, a numberof viruses have been used as gene transfer vectors, includingpapovaviruses, such as SV40, vaccinia virus, herpes viruses, includingHSV and EBV, and retroviruses. Many gene therapy protocols in the priorart have used disabled murine retroviruses. A variety of adenovirus andadeno-associated viral vectors have been developed. Alternatives toviral vectors include transfer mediated by liposomes and direct DNAuptake and receptor-mediated DNA transfer.

Expression of polynucleotides or polypeptides encoded by or derived fromaxotrophin is suitably under the control of inducible regulatoryelements, in which case the regulatory sequences of the endogenous genemay be replaced by homologous recombination. Gene targeting may be usedto replace a gene's existing regulatory region with a regulatorysequence isolated from a different gene or a novel regulatory sequencesynthesized by genetic engineering methods. Such regulatory sequencesmay be comprised of promoters, enhancers, scaffold-attachment regions,negative regulatory elements, transcriptional initiation sites,regulatory protein binding sites or combinations of said sequences.Alternatively, sequences which affect the structure or stability of theRNA or protein produced may be replaced, removed, added, or otherwisemodified by targeting. These sequences include polyadenylation signals,mRNA stability elements, splice sites, leader sequences for enhancing ormodifying transport or secretion properties of the protein, or othersequences which alter or improve the function or stability of protein orRNA molecules.

Other methods inhibiting expression of a polypeptide include theintroduction of antisense molecules to the polynucleotides of thepresent invention, their complements, their transcribed RNA sequences,or translated products of RNA by methods known in the art. Further, thepolypeptides of the present invention can be inhibited by using targeteddeletion methods, or the insertion of a negative regulatory element suchas a silencer, which is tissue specific. “Gene silencing” technology isdisclosed by Fire et al in EP-A-1042462 and Nature Vol 391 pp 806 to811, “Potent and specific genetic interference by double stranded RNA inC elegans”.

The term “isolated” as used herein refers to a polynucleotide orpolypeptide separated from at least one other component (for example,polynucleotide or polypeptide) present with the polynucleotide orpolypeptide in its natural source. In one embodiment, the polynucleotideor polypeptide is found in the presence of (if anything) only a solvent,buffer, ion, or other component normally present in a solution of thesame. The terms “isolated” and “purified” do not encompasspolynucleotides or polypeptides present in their natural source.

The term “degenerative variant” as used herein includes nucleotidesequences that differ from the sequence according to the invention butdue to the degeneracy encode an identical polypeptide sequence or asequence having at least 75% and preferably at least 90% sequenceidentity thereto.

A collection of sequence information for axotrophin or identifyinginformation for it can be provided on a polynucleotide array. In oneembodiment, segments of sequence information are provided on apolynucleotide array to detect the polynucleotide that containsaxotrophin or an axotrophin segment. The array can be designed to detectfull-match or mismatch to axotrophin. The collection can also beprovided in a computer-readable format.

The invention further provides cells genetically engineered to containaxotrophin or a vector according to the invention as described herein.Suitably the cells according to the invention, preferably host cells,have been transformed or transfected with axotrophin or anotherpolynucleotide of the invention to express axotrophin or apolynucleotide or polypeptide sequence encoded by or derived fromaxotrophin. Known transformation, transfection or infection methods maybe employed.

Systems for cloning and expression of a polynucleotide or polypeptide ina variety of different cells are known. Suitable host cells includebacteria, eukaryotic cells such as mammalian and yeast, and baculovirussystems. Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary cells, HeLacells, baby hamster kidney cells, COS cells and many others. A common,preferred bacterial host is E. coli.

A still further aspect provides a method which includes introducing thepolynucleotide into a host cell. The introduction, which may(particularly for in vitro introduction) be generally referred towithout limitation as a transformation, may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, forexample vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and transfection using bacteriophage. As an alternative,direct injection of the polynucleotide could be employed. Marker genessuch as antibiotic resistance or sensitivity genes may be used inidentifying clones containing polynucleotide of interest, as is wellknown in the art.

The introduction may be followed by causing or allowing expression fromthe polynucleotide, for example by culturing host cells (which mayinclude cells actually transformed although more likely the cells willbe descendants of the transformed cells) under conditions for expressionof the gene, so that the encoded peptide or polypeptide is produced. Ifthe peptide or polypeptide is expressed coupled to an appropriate signalleader peptide it may be secreted from the cell into the culture medium.Following production by expression, a peptide or polypeptide may beisolated and/or purified from the host cell and/or culture medium, asthe case may be, and subsequently used as desired, for example in theformulation of a composition

Suitably the polynucleotides of axotrophin expressed in cells in vivoare in operative association with a regulatory sequence heterologous tothe host cell which drives expression of the polynucleotides in thecell. These methods can be used to increase or decrease the expressionof the polynucleotides of the present invention. The invention alsorelates to methods for producing axotrophin polypeptide comprisinggrowing a culture of cells of the invention in a suitable culture mediumunder conditions permitting expression of the desired polypeptide, andpurifying the polypeptide from the culture or from the host cells.Preferred embodiments include those in which the protein produced bysuch process is a mature form of the protein and any other polypeptidesthat retain any functional activity of the mature protein. In apreferred embodiment, a polypeptide encoded by or derived fromaxotrophin is used to generate an antibody that specifically binds thepolypeptide. Such antibodies, particularly monoclonal antibodies, areuseful for detecting or quantitating the polypeptide in tissueespecially for immune diagnostic purposes. Polypeptides of the inventionmay be produced in whole or part by recombinant means but may bechemically synthesized

Such a method may comprise bringing a population of antibody moleculesinto contact with axotrophin or a polynucleotide or polypeptide encodedby or derived from axotrophin and selecting one or more antibodymolecules of the population able to bind and/or affect the activity ofthe polypeptide or polynucleotide.

Antibody molecules may routinely be obtained using technologies such asphage display, by-passing direct involvement of an animal's immunesystem. Instead of or as well as immunising an animal, a method ofobtaining antibody molecules as disclosed may involve displaying thepopulation of antibody molecules on the surface of bacteriophageparticles, each particle containing polynucleotide encoding the antibodymolecule displayed on its surface, Polynucleotide may be taken from abacteriophage particle displaying an antibody molecule able to bind apeptide or peptides of interest, for manipulation and/or use inproduction of the encoded antibody molecule or a derivative thereof (forexample a fusion protein, a molecule including a constant region orother amino acids, and so on). Instead of using bacteriophage fordisplay (as for example in WO92/01047), ribosomes or polysomes may beused, for example as disclosed in U.S. Pat. No. 5,543,768, U.S. Pat. No.5,658,754, WO95/11922.

A peptide or peptides may be administered to a non-human mammal to bringthem into contact with a population of antibody molecules produced bythe mammal's immune system, then one or more antibody molecules able tobind the peptide or peptides may be taken from the mammal, or cellsproducing such antibody molecules may be taken from the mammal. Themammal may be sacrificed.

If cells are taken from the mammal, such cells may be used to producethe desired antibody molecules, or descendants or derivative cell linesmay be used. Such descendants or derivatives in particular may includehybridoma cells.

Antibody molecules may be provided in isolated form, either individuallyor in a mixture. A plurality of antibody molecules may be provided inisolated form.

Preferred antibodies according to the invention are isolated, in thesense of being free from contaminants such as antibodies able to bindother polypeptides and/or free of serum components. Monoclonalantibodies are preferred for some purposes, though polyclonal antibodiesare within the scope of the present invention.

Antibodies useful in accordance with the present invention may bemodified in a number of ways. Indeed the term “antibody molecule” shouldbe construed as covering antibody fragments and derivatives comprisingan antibody antigen-binding domain enabling it to bind an antigen orepitope. Example antibody fragments, capable of binding an antigen orother binding partner are the Fab fragment consisting of the VL, VH, Cland CH1 domains; the Fd fragment consisting of the VH and CH1 domains;the Fv fragment consisting of the VL and VH domains of a single arm ofan antibody; the dAb fragment which consists of a VH domain; isolatedCDR regions and F(ab′) 2 fragments, a bivalent fragment including twoFab fragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

Cells may be cultured ex vivo in the presence of proteins orpolynucleotides encoded by or derived from axotrophin in order togenerate a desired immune response for example immunosuppression forsubsequent reintroduction in viva to allow introduction of immunogenicbiological material. In other uses, prevention of the expression orinhibiting the activity of axotrophin may be desirable so as to augmentaggressive immune activity against antigens. Antisense therapy or genetherapy may suitably be employed to negatively regulate the expressionof polypeptides or polynucleotides encoded by or derived fromaxotrophin.

Modification of cells or tissues to permit, increase or decreaseexpression of endogenous axotrophin polypeptide to provide increasedpolypeptide expression by replacing in whole or part the naturallyoccurring promoter with a heterologous promoter so that the cellsexpress the protein at higher levels or show induced expression inresponse to pharmaceutical compounds.

In a further aspect, the invention provides for manipulating, forexample enhancing production of autologous or other stem cells orprecursor cells and/or immune cells ex vivo by introduction to the cellof axotrophin or a polynucleotide or polypeptide encoded by or derivedfrom axotrophin, The cells are manipulated prior to in vivo delivery fortherapeutic purpose, particularly for regulating the immune response.

In a preferred embodiment, lymphocytes from an individual may becultured ex vivo in the presence of one or more specific differentiationfactors (for example target antigen for a given T cell receptor (“TCR”)and the response to that antigen adapted, modified or qualified to beregulated for tolerance or to be aggressive to the antigen, using up ofdown regulation of polypeptide or polynucleotide encoded by or derivedfrom axotrophin. The ex vivo derived differentiated clones may bepropagated and may be used to treat the recipient, especially theoriginal donor, to regulate the immune response. For example; arecipient may be rendered specifically tolerant to a foreign organallograft prior to receiving the organ graft itself.

The modulation or inducing of an immune response in the methods of thepresent invention may be provided by polypeptide or polynucleotideencoded by or derived from axotrophin, analogs including fragments andfusion proteins, antibodies and other binding proteins and chemicalcompounds that directly inhibit or activate the polypeptides ofaxotrophin activity in the immune response.

Polynucleotide molecules and vectors according to the present inventionmay be provided in isolated and/or purified form, for example insubstantially pure or homogeneous form. The term “isolate” may be usedto reflect all these possibilities.

A peptide, polypeptide, antibody, polynucleotide or other molecule oragent for use in accordance with the present invention may be formulatedinto a composition, and is useful in pharmaceutical contexts.

The present invention also relates to a composition containing isolatedaxotrophin or a polypeptide or polynucleotide encoded by or derived fromaxotrophin and a pharmaceutically acceptable diluent, carrier orexcipient which is suitably non-toxic and should not interfere with theefficacy of the active ingredient. The precise nature of the carrier orother material may depend on the route of administration, for exampleoral, intravenous, cutaneous or subcutaneous, nasal, intramuscular,intraperitoneal routes.

The diluent, carrier or excipient may be in the form of a gel, an oil ora liposome and, independently, preferably comprises a hydrophilicmaterial, for example water. The precise nature of the carrier or othermaterial may depend on the route of administration, for example oral,intravenous, cutaneous or subcutaneous, nasal, intramuscular,intraperitoneal routes.

Compositions for oral administration may be in tablet, capsule, powderor liquid form. A tablet may include a solid carrier such as gelatin oran adjuvant. Liquid pharmaceutical compositions generally include aliquid carrier such as water, petroleum, animal or vegetable ails,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, the activeingredient will suitably be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included, as required.

The composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated and the availability of alternative oradditional treatments.

In the present invention, a composition may be administered to anindividual, particularly human or other primate. Administration may beto a human or another mammal, for example rodent such as mouse, rat orhamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey, dogor cat. Delivery to a non-human mammal need not be for a therapeuticpurpose, but may be for use in an experimental context, for instance ininvestigation of mechanisms of immune responses to an antigen ofinterest, for example protection against cancers, pathogens and so on.

This invention is particularly useful for screening chemical compoundsby using axotrophin or polynucleotides or polypeptides encoded by orderived from axotrophin or binding fragments thereof in drug screeningtechniques.

The invention provides a method of screening chemical compoundscomprising contacting a test sample containing one or more chemicalcompounds to be screened with a binder selected from axotrophin, apolynucleotide or polypeptide encoded by or derived from axotrophin andfragment of such polynucleotide or polypeptide and determining whetherthe chemical compound has bound to the binder.

The binder may be in any suitable form including a vector, cell orcomposition and utilized in known ways of screening for chemicalcompounds.

The polypeptides polynucleotides or fragments employed in such a testmay either be free in solution, affixed to a solid support, borne on acell surface or located intracellularly. One method of drug screeningutilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant polynucleotides expressing the axotrophinpolypeptide or a fragment thereof. Chemical compounds may be screenedagainst such transformed cells in competitive binding assays. Suchcells, either in viable or fixed form, may be used for binding assays ina known manner.

Isolated proteins and polynucleotides of axotrophin may be used toobtain and identify agents which bind to a polypeptide encoded by orderived from an open reading frame (“ORF”) corresponding to axotrophinor bind to a specific domain of the polypeptide encoded by or derivedfrom axotrophin.

The invention provides a screening method for identifying an agent whichbinds to axotrophin or a polypeptide or polynucleotide encoded by orderived from axotrophin comprising:

-   -   (a) contacting an agent with axotrophin or a or polynucleotide        polypeptide encoded by or derived from axotrophin;    -   (b) determining whether the agent binds to the said        polynucleotide or polypeptide; and    -   (c) detecting the formation of a complex, formed between the        agent and the said polynucleotide or polypeptide such that if a        complex is formed, the agent is detected.

In a preferred screening method the compound is contacted with apolypeptide or polynucleotide of axotrophin in a cell for a timesufficient to form a polypeptide complex of the compound with thepolypeptide or polynucleotide, wherein the complex drives expression ofa receptor gene sequence in the cell, and detecting the complex bydetecting reporter gene sequence expression.

The invention also provides a kit comprising axotrophin or apolynucleotide probes and/or monoclonal antibodies, and optionallyquantitative standards, for carrying out methods of the invention.

The present invention further provides a diagnostic method to identifythe presence or expression of axotrophin or a polypeptide orpolynucleotide encoding axotrophin in a test sample, using apolynucleotide probe or antibodies to axotrophin, optionally conjugatedor otherwise associated with a suitable label.

The invention provides a diagnostic method for detecting axotrophin or apolynucleotide or polypeptide encoded by or derived from axotrophincomprising:

(a) contacting a sample to be tested for the presence of apolynucleotide or polypeptide encoded by or derived from axotrophin witha compound that binds to a polynucleotide or polypeptide encoded by orderived from axotrophin;

(b) determining whether the compound binds to a component of the sample;and (c) detecting the formation of a complex, formed between the agentand the protein or polynucleotide and such that if a complex is formed,the polypeptide or polynucleotide is detected.

Preferably the diagnostic method comprises contacting a sample understringent hybridization conditions with polynucleotide primers thatanneal to a polynucleotide of axotrophin and amplifying annealedpolynucleotides, so that if a polynucleotide is amplified, apolynucleotide of axotrophin is detected in the sample.

In a preferred embodiment, the diagnostic method for assessing theimmune response of an individual comprises obtaining a test sample fromthe individual, for example blood, incubating the test sample with oneor more of the antibodies or one or more of a polynucleotide probes foraxotrophin or a polynucleotide or polypeptide encoded or derived fromaxotrophin and assaying for binding of the polynucleotide probes orantibodies to components within the test sample.

Assays according to embodiments of the present invention may employELISA, Western blot, immunohistochemistry, identification of the effectsof drugs on the immune response in terms of induced bias towardsregulatory tolerance, anergy or deletion, versus rejection and any othersuitable technique available in the art.

Tests may be carried out on preparations containing cDNA and/or mRNA.RNA is more difficult to manipulate than DNA because of the wide-spreadoccurrence of RN′ases, which is one reason why CDNA analysis may beperformed.

However, since it will not generally be time or labour-efficient tosequence all polynucleotide in a test sample or even the whole gene ofinterest, a specific amplification reaction such as PCR using one ormore pairs of primers may be employed to amplify the region of interestin the polynucleotide if present in the sample. This may be donequantitatively, allowing for determination of the amount of axotrophinor a polypeptide or polynucleotide encoded by or derived from axotrophinin the test sample.

Polynucleotide may be screened using a specific probe Such a probecorresponds in sequence to a region of the relevant gene, or itscomplement Under suitably stringent conditions, specific hybridisationof such a probe to test polynucleotide is indicative of the presence ofthe polynucleotide molecule of interest, and again this may bequantitated to provide an indication of the amount of suchpolynucleotide molecule in the test sample.

Specific oligonucleotide primers may similarly be used in PCR tospecifically amplify particular sequences if present in a test sample.

A method may include hybridisation of one or more (for example two)probes or primers to target polynucleotide. Where the polynucleotide isdouble-stranded DNA (e.g. cDNA), hybridisation will generally bepreceded by denaturation to produce single-stranded DNA, Thehybridisation may be as part of a PCR procedure, or as part of a probingprocedure not involving PCR. A screening procedure, chosen from the manyavailable to those skilled in the art, is used to identify successfulhybridisation events and may allow for quantitation of the amount ofpolynucleotide present in the original sample.

Binding of a probe to target polynucleotide (for example DNA) may bemeasured using any of a variety of techniques at the disposal of thoseskilled in the art. For instance, probes may be radioactively,fluorescently or enzymatically labeled. Probing may employ a standardblotting technique.

A test sample of polynucleotide may be provided for example byextracting polynucleotide from cells such as spleen cells or biologicaltissues or fluids, urine, saliva, faeces, a buccal swab, biopsy orblood.

A test sample may be tested for the presence of a binding partner for aspecific binding member such as an antibody molecule (or mixture ofantibodies), specific for the polypeptide or polypeptide of interest.The sample may be tested by being contacted with a specific bindingmember such as an antibody molecule under appropriate conditions forspecific binding, before binding is determined, for instance using areporter system as discussed. Where a panel of antibodies is used,different reporting labels may be employed for each antibody so thatbinding of each can be determined.

A specific binding member such as an antibody molecule may be used toisolate and/or purify its binding partner polypeptide from a testsample, to allow for sequence and/or biochemical analysis of thepolypeptide to determine whether it has the sequence and/or propertiesof axotrophin or a polypeptide or polynucleotide encoded by or derivedfrom axotrophin. Amino acid sequencing is routine in the art usingautomated sequencing machines.

A test sample containing one or more polypeptides may be provided forexample as a crude or partially purified cell or cell lysatepreparation, for example using tissues or cells, such as from the spleenor a bodily fluid, preferably blood.

Other tests may involve the use of blood or spleen cells taken from atest animal, individual, subject or patient, and ex vivo challenge ofthe cells with antigen to determine the presence or absence of anaggressive or tolerant response to the antigen.

Suitable probes may, for example, be used to determine whether specificmRNA molecules are present in a cell or tissue or to isolate similarpolynucleotide sequences from chromosomal DNA, for example as describedby Walsh et al. (Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).They may be labeled by nick translation, Kienow fill-in reaction, PCR,or other methods known in the art. Suitable probes, their preparationand/or labeling are elaborated in Sambrook, J. et al., 1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; orAusubel, F. M. et al., 1989, Current Protocols in Molecular Biology,John Wiley & Sons, New York N.Y. All documents mentioned anywhere inthis specification are incorporated by reference. The invention isillustrated by the following non-limiting Examples and accompanyingFigures.

EXAMPLE 1

Transplantation Tolerance: Gene Expression Profiles Comparingallo-tolerance Versus allo-jection

In mice, infectious regulatory tolerance is inducible by CD4/CD8blockade in recipients of vascularised heart grafts. Once established,this transplantation tolerance is robust and isolated “tolerant” spleencells show powerful immune regulatory properties, being able to imposedonor-specific allo-tolerance upon fully immune competent naiverecipients. Using ^(BALB/c-tolerant)CBA (H-2^(k)) mice, we analysedspleen cell responses to donor (BALB/c [H-2^(d)]) antigen at a series oftime points and in comparison with an identical ex vivo series of^(BALB/C-rejected)CBA spleen cells. The key feature of rejection wasrapid Interferon gamma release. In contrast, Interferon gamma intolerance was low and less than that released in response to third partyantigen (C57Bl10 [H-2^(b)]). Positive markers of primed tolerance werehigh expression of STAT3 and c-kit, and release of LIF. Here we presenta compound comparison of four gene arrays (tolerance versus rejection,at 48h, and at 123h) where a relatively small number of differentiallyexpressed genes occurred. In rejection, there was a strong progressiveamplification of Interferon gamma and granzyme B mRNAs. In tolerance,both Emk and axotrophin were upregulated at 123h. Mice lacking Emkdevelop auto-immune disease (Hurov et al, Mol Cell Biol, 2001). Micelacking axotrophin show abnormal axonal migration during development.Taken together, our results suggest a link between developmentalregulation and immune regulation, and highlight a possible role foraxotrophin in regulatory tolerance.

Materials and Methods

Generation of ^(BALB/c-primed) CBA Mice.

CBA mice (H2^(k)) of 10-12 weeks of age received a fully mismatched,vascularised BALB/c (H2^(d)) heart graft to the neck, using thetechnique described by Chen, [Chen Z. K., Cobbold, S. P., Waldmann, H. &Metcalfe, S. M. Amplification of natural regulatory immune mechanismsfor transplantation tolerance. Transplantation 62, 1200-1206 (1996)].Tolerance was generated by a 21 day course of alternate day therapyusing blocking mAbs to CD4 and CO8 as previously described [Chen, Z. K,,Cobbold, S. P., Waldmann, H, & Metcalfe, S. M. Amplification of naturalregulatory immune mechanisms for transplantation tolerance.Transplantation 62, 1200-1206 (1996)]. ^(BALB/c-tolerant)CBA spleencells from tolerant recipients were isolated at least 100d aftergrafting for ex vivo analyses. For comparison, untreated CBA mice, 10-12weeks of age, were grafted either with BALB/c tail skin which rejectedby day 10, or with a BALB/c heart which rejected on day 7. The^(BALB/c-rejected)CBA spleen cells were collected at 14d for ex vivoanalyses. All procedures were carried out according to Home Officelicence under the Animals (Scientific Procedures) Act 1986, UK.

Ex vivo Cultures

Culture conditions have been described in detail elsewhere [Metcalfe, S.M. & Moffatt-Bruce, S. D. An ex vivo model of tolerance versusrejection: Comparison of STAT1, STAT4, STAT5 and STAT6. Clin. Chem. andLab. Med. 38, 1195-1199 (2000)]. Briefly, responder spleen cells wereobtained from either ^(BALB/c-tolerant)CBA, or ^(BALB/c-rejected)CBA,mice, and the tolerant and rejected cell populations were stimulated exvivo by irradiated BALB/c spleen cells (donor antigen), using 4×10⁷responders to 6×10⁷ stimulators in a total of 10 ml growth mediumsupplemented with 10% FCS. After 48h, one flask each of tolerant andrejected spleen cells were removed for total RNA preparation. A secondpair of flasks (one tolerant, one rejected) were boosted with a further7×10⁷ stimulator spleen cells at 120 h, and then harvested at 123 h. Atharvest, cells were collected onto ice, with any adherent cells beingincluded following brief treatment with 0.25% trypsin. Afterresuspending the cells to homogeneity, a 1.5 ml aliquot was removed forRNA extraction. After washing in ice cold 0.1% BSA/PBS, the cells werecollected into sterile 15 ml Falcon centrifuge tubes and pelleted at1600rcf for 5 min at +4° C. Supernatant was discarded and the tube wipedclear of supernatant residue prior to resuspending the cells inpre-cooled Trizol reagent, vortexed, and then immediately stored at −80°C. One ml Trizol was used per 6×10⁻⁶ cells.

RNA Isolation.

Samples were brought to room temperature and kept for 10 minutes beforeaddition of 1 ml chloroform and vortexing to an emulsion. After 15 minthe samples were centrifuged at 1600rcf for 10 min at 40C. The upperphase was transferred to RNA-ase-free Eppendorff tubes in 400 μlaliquots and an equal volume of isopropanol added. After gentle mixingand standing for 15 min, the samples were centrifuged at 13,000 g at 40Cfor 10 min. The supernatant was removed and discarded. The RNA pelletwas washed in 350 μl of 75 ethanol and sedimented at 7500 g for 5 min at40C. The supernatant was aspirated and the pellet air dried for 20 min.The aliquoted RNA pellets were collected together for each sample bydissolving and serial transfer of 50 μl DH₂O; a second 50 μl was used toserially collect washings from each tube, giving a final total samplevolume of 100 μl in DH₂O. This was stored at −80° C. until transfer tothe MRC HGRC at Hinxton Hall for customer service preparation of cRNAand array using Affymetrix U74 chips by standard methodologies.

Gene Array.

Analyses of the combined arrays was prepared using dChip software [Wong,C.U.W.H., PNAS USA, 98, 31, 2001].

RESULTS

Combined 48 h and 123 h arrays of the matched tolerant and rejectedsamples pairs gave 129 genes showing differential expression. Toidentify those genes that showed a biased expression in eithertolerance, or in rejection, the results were ranked in three ways: thosegenes showing a positive shift from 48 h to 123 h (Table 1); those geneswith high expression at 123 h (Table 2); and those genes (tolerant) thatshowed a positive shift, whilst the rejection counterpart showed anegative shift from 48 h to 123 h (Table 3).

Of the genes that increased in expression from 48 h to 123 h, 10 were inthe tolerant cultures with increases ranging from 1.71 fold to 4.00fold. Expression of the same genes in the rejection response showedeither no increase in expression or a decrease in expression (Table1(a)). Of particular note was axotrophin, a newly discovered stem cellgene; cyclin B2, associated with the cell cycle and cellular migration;histone H2A-X that may play a role in chromatin remodeling; and ELKLmotif kinase, also known as Erk, required to regulate the immuneresponse and protect against auto-immunity. Table 1 (b) shows the 5genes that increased in expression in rejection. Again this increase wasspecific to rejection, with the exception of granzyme B with a twofoldincrease in both tolerance and rejection; however, the actual levels ofgranzyme B mRNA were six times greater in rejection than in tolerance.The 12-fold increase in Interferon gamma mRNA in rejection was in accordwith our previous findings of high Interferon gamma protein release inthese cultures.

Of those genes that showed high expression at 123 h, within the contextof the four arrays, 15 were in the tolerant set (Table 2(a)) andincluded axotrophin. In rejection, 13 genes are ranked in order ofexpression level in Table 2(b) with granzyme B and Interferon gammabeing the highest. This analytical approach therefore showed correlationwith phenotype with respect to granzyme B and Interferon gamma, andagain placed axotrophin as being associated with tolerance, although theactual expression level was not great A further analysis was made,identifying those genes that showed increased expression in tolerancewhilst showing a decreased expression in rejection (Table 3). Thisrevealed Histone H2A-X, involved in chromatin structure and remodeling;ELKL motif kinase; splicing factor 3b subunit 1 (SF3b-155), acting aspart of the mRNA splicing complex and probably involved in exon removal;and cyclin B2, a regulator of the cell cycle and also involved incellular migration when complexed with cdc2. TABLE 1a and 1b Genesshowing increased expression {48 h versus 123 h) Tolerance: Rejection:Accession Fold Fold Gene Number increase increase TOLERANCE Dualspecificity phosphatase 1 X61940 4.00 0.99 BCL2-like 11 AA796690 3.111.15 Axotrophin* AW212859 2.9 1.00 H2A histone family, member X M339882.22 0.46 Interferon stimulated protein AW122677 2.21 0.95 (20 kDa)Chemokine (C-C) receptor 6 AJ222714 2.02 0.95 Cyclin B2 X66032 2.01 0.59Paneth cell enhanced expression U37351 2.0 0.98 Splicing factor 3b,sub-unit 1, A1844532 1.93 0.59 155 kDa ELKL motif kinase** X70764 1.710.63 REJECTION Interferon gamma K00083 0.69 11.98 Glutaryl CoAdehydrogenase U18992 1.20 5.10 CD3 antigen, gamma polypeptide M182281.23 3.22 Interleukin 1 receptor antagonist L32838 1.00 2.57 Granzyme BM12302 2.07 2.52

TABLE 2a and 2b Genes showing high expression at 123 h within thecontext of the four arrays Expression Accession level @ Gene Number 123h TOLERANCE □-2 microglobulin X01838 9047  Ring Finger protein 10AB026621 4127  CD53 antigen X97227 3927  Guanylate nuceotide bindingprotein 1 M55544 1005  Spermidine spermineN1 acyl transferase L102441002  Glycoprotein 49A M65027 975 Chemokine (C-C) receptor 6 AJ222714 972) BCL2-like 11 AA796690 752 Paneth cell enhanced expression U37351753 EST AW047461 744 Chemokine (C-C motif) ligand 9 C-U49513 593 ESTA1060627 562 Dual specificity phosphatase 1 X61940 536 ExpressedSequence AU021774 A1854141 438 Axotrophin* AW212859 416 REJECTIONGranzyme B M12302 6766  Interferon gamma K00083 3103)  Metallothionein 2KO2236 1952  Lectin, galactose binding, soluble 1 X15986 1887  RNAbinding motif protein 3 AB016424 1725  Acidic nuclear Phosphoprotein 32family, A1842771 1665  member B Glutaryl-Coenzyme A dehydrogenase U189921350  STAT3 U08378 1026  STAT5A AJ237939 988 Calcylcin X66449 856 CD3antigen pyrophosphate M18228 517 IL1 receptor antagonist L38838 511 ExpSequence AU044919 X67210 356

TABLE 3 Genes showing increases in expression in tolerance and decreasedexpression in rejection Accession Gene Number Gene description H2Ahistone family, M33988 Chromatin remodeling (Bassing; member X Bruno)ELKL motif kinase** X70764 Immune regulation ((Hurov) Splicing factor3b, A1844532 RNA splicing, intron removal (Horie) subunit 1, 155 kDaCyclin B2 X66032 Cell cycle; cell migration (Manes)

EXAMPLE 2

The Stem Cell Gene axot is Associated with Regulation of LIF andMitogenic Activation of T Lymphocytes.

Control of “stemness”¹ for self-renewal of stem cells, versus theirdifferentiation during organogenesis, is fundamental to the new field ofregenerative medicine, Leukaemia inhibitory factor (LIF) is critical tothis control, acting as a suppressor of stem cell differentiation^(2,3). The finding that both LIF and axot, a novel stem cellgene^(1,4), are linked also to immune tolerance suggests a relationshipbetween sternness and immunity. To explore this relationship we haveasked if immune cells from axot^(−/−) mice differ from those ofaxot^(+/+) littermates. We discovered (i) that presence of axotrophin isinvolved in damping down proliferation of T, but not B, lymphocytes;(ii) that lack of axotrophin leads to excessive release of T cellcytokines: and (iii) an axot gene-dose dependent suppression of LIF.This is the first evidence that fate determination mediated by LIF maybelinked to axotrophin, and demonstrates commonalities between sternnessand immune tolerance that may favour acceptance of implanted stem cellallo-grafts for therapeutic tissue regeneration.

Fate determination in stem cells is a critical feature in development,providing q balance between pluripotent self-renewal versusdifferentiated function within the whole organism. In regenerativemedicine, understanding the molecular basis of fate determination ofstem cells is important if they are to be used successfully in thetreatment of disease. Fate determination pathways also play a key rolein the immune system, where reactivity is finely tuned to ensureprotective tolerance towards self tissues whilst simultaneously beingcapable of aggressive attack towards foreign pathogens. Although theregulatory tolerance pathway is little understood, the recentdemonstration that a single gene, foxp3, is able to orchestrate thedifferentiation of naive CD4+T cells into regulatory T cells(Treg)^(5,6,7) implies the existence of “master” switches for fatedetermination in immunity. We have recently discovered features ofimmune tolerance that are common to regulation of stem cell fate,raising two important questions: do “stemness” signals play a role inautoimmunity by suppressing terminal differentiation of immune effectorcells? Do allogeneic stem cells bias the alto-immune response towardsallo-tolerance, by signaling for “stemness”, so favouring successfultherapeutic engraftment? This paper describes how we discovered thataxotrophin, expressed in embryonic, neuronal, and haematopoletic stemcells¹, is not only involved in regulation of T lymphocyte reactivity,but also in regulation of LIF, thereby providing a novel concept ofimmunoregulation.

The molecular events associated with immune tolerance, versus immuneaggression, have been compared in previous studies using an ex vivomodel⁸. This is derived from mice where fully mismatched heart grafts,normally rejected by day 7, become accepted indefinitely after shortterm blockade of CD4 and CD8 (ref. 9). Once established, thistransplantation tolerance is self-perpetuating and isolated “tolerant”spleen cells show powerful immune regulatory properties, being able toimpose donor-specific alto-tolerance when infused into fully immunecompetent naive recipients. We characterised the ex vivo responses ofthe tolerant spleen cells, versus spleen cells from mice that had beenprimed to reject the same donor-type and the key features of rejectionwere rapid interferon gamma release and strongly amplified expression ofgenes encoding Interferon gamma and granzyme B. In marked contrast,tolerance showed features in common with sternness, these being therelease of LIF and increases in c-kit (the receptor for stem cell factor(SCF)) and in STAT3 (signal transducer and activator of transcription 3,responsive to both SCF and LIF activity). We found that the relationshipbetween LIF and tolerance was also evident in cloned Treg, showing highlevels of LIF release in contrast to Th1 and Th2 clones. At the genelevel, tolerance was associated with strong induction of a newlydiscovered stem cell gene, axot (Genbank accession number AF155739). Totest of our hypothesis that sternness and tolerance are linked, we haveasked if axotrophin influences immune responsiveness.

We first looked at lymphocyte responsiveness to mitogen. Axot null(axot^(−/−) mice were compared to littermates that expressed either oneaxot allele (heterozygous, axot^(+/−)) or both alleles (wild-type;axot^(+/+)). Whole cell populations were freshly isolated from thespleen and we measured mitogenic activation using either concanavalin A(conA) as a T cell mitogen, or lipopolysaccharde (LPS) as a B cellmitogen. We also looked for any kinetic effects on responsiveness bycomparing DNA synthesis at 48 h and at 72 h. Since activated lymphocytesshow a synchronised entry into the cell cycle, with S phase peaking at48 h (ref. 10), we reasoned that a consistent reduction in DNA synthesisin the axot null cells, compared to the axot^(+/+) cells, would indicatea loss of mitogenic responsiveness due to lack of axotrophin. However,the level of T cell proliferation showed a marked increase in the axotnull cells when compared to wild-type cells. This was not caused byaltered kinetics since the axot-related differentials were similar atboth 48 h and 72 h (FIG. 1 a, FIG. 1 b). Therefore axotrophin appearedto be repressing the proliferative response of T cells. Moreover, sincethe heterozygous axot^(+/−) T cells showed intermediatehyper-proliferation, the repression appeared sensitive to axot genedose. In marked contrast to the T cells, B lymphocyte proliferation wasnot significantly altered by axotrophin (FIG. 1 c, FIG. 1 d). Weconcluded that axotrophin plays a role in damping down T, but not B,lymphocyte proliferation following mitogenic stimulation. No spontaneousmitogenesis occurred in cultures of axot^(+/+), axot^(+/−) or axot^(−/−)spleen cells over a 7 d period.

As a further test of functionality in the axot null spleen cells, wemeasured cytokine release in response to mitogen. Lack of axotrophin wasassociated with a twofold increase in interleukin 2 (IL2) following conAtreatment, in both axot null and axot heterozygous cell cultures (FIG. 2a). This IL2 equivalence revealed that IL2 was not a limiting factor forT cell proliferation, where there had been a four-fold difference.Splenic B cells did not release IL2 (FIG. 2 b) whilst both T and B cellsreleased IL10 in response to their respective mitogens. Again, only theconA-treated cultures were affected by a lack of axotrophin, with aten-fold increase in IL10 in both axot^(+/−) and axot^(−/−) cellcultures (FIG. 2 c, FIG. 2 d). These findings show that partial or totalreduction of axotrophin results in a general increment in both IL2 andIL10 from activated T cells, but has no effect on IL10 release fromactivated B cells. Interferon gamma and IL4 were also measured andshowed a similar axot-related increment to that found for IL2 in theconA-treated cultures, as detailed in the legend to FIG. 1. LPS-treatedcultures were negative for Interferon gamma and IL4.

Unexpectedly, we found that release of LIF in response to conA wasstrongly inhibited by axotrophin and that this inhibition was gene-dosedependent (FIG. 2). There was no LIF in the LPS-treated culturesirrespective of axot genotype. Based on the relationship between LIFconcentration versus axot gene dose, we have hypothesised that gene dosecorrelates with expression levels of axotrophin. Both LIF release and Tcell proliferation would thus appear to be critically influenced byaxotrophin and our results would be in accord with inter-dependent linksbetween the three.

By analysis of phenotype and of histological structure, we looked foreffects of axotrophin on the phenotypic composition of lymphoid organs.Cell sub-populations were identified by FACS analysis as follows: cellsexpressing the T cell markers CD3, CD4 and CD8; the B cell marker CD19:;the activation marker of T cells and of regulatory tolerant T cells,CD25; and markers of dendritic cells, CD205 and DC33D1. None of thesemarkers showed differential expression between the axot^(+/+),axot^(+/−), and axot^(−/−) littermates (FIG. 3). Similarly, histologicalassessment of the spleen and thymus showed no significant differencesbetween the three axot genotypes.

Fate determination is controlled by genetic programmes that are alteredby changing the nature and frequency of cytokine interactions within themicroenvironment, both for totipotent and pluripotent stem cells, andfor the differentiation of precursor cells. LIF Is a key determinant ofself-renewal of stem cells¹¹ in addition to being a neuropoieticcytokine¹². Having shown that axotrophin may act as a negative regulatorof LIF, at least in activated T cells, we suggest that LIF expression isfunctionally coupled to axotrophin expression, with axotrophin playing arole in co-ordinating the positive and negative regulation of LIFrelease. This would place axotrophin as a potential regulator of fatedetermination via LIF. The molecular function of axotrophin has yet tobe determined and how axotrophin might influence LIF release is unknown.Future work will include exploration of this relationship, looking foreffects of axotrophin on LIF gene expression¹³, and on regulation ofLIF-induced signaling through the LIF-R/gp130 complex^(14,15,16,17).

As a working model we propose that LiF activity, regulated byaxotrophin, is associated with immune tolerance. LIF may guide naive Tcells towards a relatively undifferentiated, non-aggressive phenotype inresponse to presented antigen, where the circumstances of presentationinitiate the tolerogenic LIF activity, either directly or indirectly(e.g. antigen presentation by immature or regulatory dendriticcells^(18,19) and associated vitamin D activity²⁰; or reduced T cellresponsiveness due to altered function of CD4/CD8 (ref. 9) or CD28 (ref.21)). Thereafter, epigenetic changes, including expression of foxp3 andROG¹⁸, and induction of Id transcription factors²², would stabilise thetolerant phenotype for inheritable Treg activity. A link between stemcell biology and regulatory immune tolerance has direct relevance totherapeutic intervention of immune-related diseases and toimmunosuppressive treatment of organ transplant recipients. The workalso has major implications for use of stem cells for regenerativemedicine, since the properties we have discovered may enhance successfuloutcome of implanted stem cells in patients.

In summary, we have discovered that axotrophin represses T lymphocyteproliferative responsiveness in adult mice and that axotrophin is ableto act as a negative regulator of LIF, implying that axotrophin actsthrough LIF to regulate T cells.

METHODS

Mice

Gene trap insertion was used to generate axot null BALB/C mice andlittermates from heterozygous parents were genotypes by PCR analysis ofgenomic DNA to identify axot^(+/+), axot^(+/−), and axot^(−/−) pups asdetailed previously. Spleen, thymus and lymph node were obtained from 5m old littermates and kept on ice prior to cell preparation for theanalyses described below. The lymph node tissue yielded very few cellsand was discarded. Spleen and thymus from axot^(+/+), axot^(+/−), andaxot^(−/−littermates) were also taken for histology. These were bisectedand fixed in 70% ethanol. Fixed tissues were embedded in paraffin blocksand sectioned, then stained with haematoxylin and eosin using standardprocedures.

Proliferation Assays

Splenocytes and thymocytes were teased out from each organ and collectedin sterile growth medium [RPMI-1640 (Gibco™ Invitrogen Co.) supplementedwith 10% FCS (Gibco™ Invitrogen Co.), 200 mM L-Glutamine, 100 U/mLPenicillin and 100 μg/mL Streptomycin (Sigma Chemical Co.)]. The cellsuspensions were washed, resuspended in growth medium and counted usinga haemocytometer.

The cells were seeded in 100 μl growth medium at 5×10⁵ nucleated cellsper well in flat bottomed 96-well Nunclon™ tissue culture plates andincubated at 37° C., 5% CO₂ for 48 h or 72 h. LPS, (Sigma Chemical Co.)at 50 μg/mL and conA (ICN Biochemicals, USA) at 10 μg/mL, were added asmitogens at time zero. All experiments were performed in triplicate.Immediately prior to harvest, supernatants were collected for ELISAanalysts and the cells were incubated for 2 hrs in pre-wan-ned GMcontaining methyl-[³H] Thymidine (TRK686, specific activity 80Ci/mmol,Amersham Biosciences) at a final concentration of 1 μCi/mL. Cells wereharvested using a Filtermate196, Packard harvester and counted using aPackard TopCount.NXT™ microplate scintillation and luminescence counter.

To determine the effect of LIF on Con A stimulation, BALB/c axot^(+/+)splenic and thymic cells were incubated in the presence of Con A (2μg/mL or 10 μg/mL) together with 500 pg/mL or 1000 pg/mL rmLIF (SantaCruz Biotechnology, SC-4378). Mitogensis was measured as describedabove. Controls included GM only, conA only, and LIF only, at therespective concentrations.

Elisa

ELISA's were performed on the 48 h culture supernatants, in 96-wellFalcon® plates using the DuoSet® ELISAS for Interferon gamma (DY4855,IL2 (DY402), IL4 (DY404), IL10 (DY417) and Quantikine®M Immunoassay forLIF (MLF00), from R&D Systems. The standard curves were established byprocessing the optical density data using Microsoft Excel software andcytokine concentrations were determined using the standard curves.

Flow Cytometry

The splenic and thymic cell suspensions were RBC depleted and washed inFACS staining solution (0.2% BSA and 0.1% sodium azide in 1×PBS) priorto being mixed with the various monoclonal antibodies detailed below,these being either directly or indirectly conjugated with Phycoerythrin(PE) or Fluorescein isothiocyanate (FITC), PE-rat anti-mouse CD19(557399), PE-hamster anti-mouse TCRα chain (553172) and rat anti-mousedendritic cell clone 33D1 (551776) were from Pharmingen. Rat anti-mouseCD205-FITC (MCA949F), mouse anti-rat IgG2a heavy chain-FITC (MCA278F)and mouse anti-rat IgG2b chain-FITC were from Serotec Ltd. while rabbitanti-mouse CD25 (IL2Rα) and goat anti-rabbit IgG (H&L)-PE (4050-89) werefrom Santa Cruz Biotechnology and Southern Biotechnology Associatesrespectively. Anti CD4 (YTS177.9.6) and anti CD8 (YTS 105.18.10) were agift from Professor Stephen Cobbold, University of Oxford. Analyses wereperformed on a Becton Dickinson FACSCalibur instrument equipped withCellQuest software.

REFERENCES

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2. Murray, P. & Edgar, D. The regulation of embryonic stem celldifferentiation by leukaemia inhibitory factor (LIF). Differentiation68, 227- 234 (2001).

3. Viswanathan, S. et al. Supplementation-dependent differences in therates of embryonic stem cell self-renewal, differentiation, andapoptosis. Biotechnol. Bioeng. 84, 505-517 (2003).

4. Baker, R. K. et al In vitro preselection of gene-trapped embryonicstem cells for characterising novel developmentally regulated genes inthe mouse. Dev Biol. 185, 201-214 (1997).

5. Hori, S. et a/, Control of regulatory T cell development by thetranscription factor Foxp3. Science 299,1057-1061 (2003).

6. Fontenot, J. D., Gavin, M. A., & Rudensky, A. Y. Foxp3 programs thedevelopment and function of CD4+CD25+ regulatory T cells. Nat. Immunol.4, 330-336 (2003).

7. Khaltri R., Cox, T., Yasayko, S. A. & Ramsdell, F. An essential rolefor Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 4, 337-342(2003).

8. Metcalfe, S. M. & Moffatt-Bruce, S. D. An ex vivo model of toleranceversus rejection: Comparison of STAT1, STAT4, STAT5 and STAT6. Clin.Chem. and Lab. Med. 38, 1195-1199 (2000)

9. Chen, Z. K., Cobbold, S. P., Waldmann, H. & Metcalfe, S. M.Amplification of natural regulatory immune mechanisms fortransplantation tolerance. Transplantation 62, 1200-1206 (1996).

10. Milner, S. M. Activation of mouse spleen cells by a single shortpulse of mitogen. Nature 268, 441-442 (1977).

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12 Patterson, P. H. Leukemia inhibitory factor, a cytokine at theinterface between neurobiology and immunology. Proc. Natl. Acad. Sci.911 7833-7835 (199).

13. Bamberger, A. M., et al. Regulation of the human leukemia inhibitoryfactor gene by ETS transcription factors. Neuroimmunomodulation 11,10-19. (2004)

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15. Cheng, J. G., Chen, J. R., Hernandez, L., Alvord, W. G. & Stewart,C. L. Dual control of LIF expression and LIF receptor function regulateStat3 activation at the onset of uterine receptivity and embryoimplantation.

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16. Takahashi, Y. et a/. SOCS3: an essential regulator of LIF receptorsignaling in trophoblast giant cell differentiation. EMBO J 22, 372-384(2003).

17. Bartoe, J. L. & Nathanson, N. M. Independent roles of SOCS-3 andSHP-2 in the regulation of neuronal gene expression by leukemiainhibitory factor. Brain Res Mol Brain Res. 107,109-119 (2002).

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20. Adorini L. Tolerogenic dendritic cells induced by vitamin D receptorligands enhance regulatory T cells inhibiting autoimmune diabetes, Ann.N. Y. Acad. Sci. 987, 258-261 (2003).

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Figure Legends

FIG. 1.

DNA Synthesis and Cytokine Release by Splenocytes from axot^(+/+),axot^(+/−), and axot^(−/−) Littermates

(a) H³-thymidine labeling of spleen cells stimulated for 48 h (upperpanels) or 72 h (lower panels) with conA (left-hand panels) or LPS(right-hand panels) DNA synthesis and standard deviation are shown aftersubtraction of the respective background controls for each genotype.Background controls were all less than 300 cpm. (b) levels of IL2 andIL10 in supernatants at 48 h after stimulation with either conA (upperpanels) or LPS (lower panels). Interferon gamma and IL4 were alsomeasured: Interferon gamma was present in the conA culture supernatantsonly, the concentrations being 538 pg/ml, 1410pg/ml, and 909 pg/mlrespectively for axot^(+/+), axot^(+/−), and axot^(−/−) cultures. IL4was also only found in the conA supernatants and was 121 pg/ml, 263pg/ml, and 92 pg/ml respectively for axot^(+/+), axot^(+/−), andaxot^(−/−) cultures. The regression analyses for goodness of fit of eachELISA were as follows, IL2, R²=0.946; IL4, R²=0.925, IL10, R²=0.939; andInterferon gammaR²=0.937.

FIG. 2.

Effect of axotrophin on LIF Release.

LIF release from spleen cells of axot^(+/+), axot^(+/−), and axot^(−/−)littermates after 48 h conA (left panel) or 48 h LPS (right panel)stimulation. The regression analyses for goodness of fit was R²=0.999.

FIG. 3.

Phenotypic Profile of Spleen and Thymus from axot^(+/+), and axot^(−/−)Mouse Littermates.

Whole populations of spleen and thymic cells were prepared, stained andanalysed as described in Materials and Methods. The FACs data ispresented in histogram format with the cut-off for negative stainingindicated by the vertical line through each data set of CD4, CD8, CD3,CD19, DC33d1, and CD25 staining. The mouse axot^(+/+) and axot^(−/−)genotypes are as indicated above each panel. Axot^(+/−) splenocytes andthymocytes were also analysed and gave the same results as those shown.CD205 staining was negative throughout.

1 . Use of axotrophin or a polypeptide or polynucleotide encoded by orderived from axotrophin to induce or to regulate, directly orindirectly, the immune response of an individual and/or ex vivo cellpopulation to an antigen.
 2. Use of axotrophin or a polypeptide orpolynucleotide encoded by or derived from axotrophin in the manufactureof a medicament to induce or to regulate, directly or indirectly, theimmune response of an individual and/or ex vivo cell population to anantigen.
 3. Use according to claim 1 or claim 2 in which the immuneresponse is in a vertebrate wherein the individual has a tissue or celltransplant.
 4. Use in the manufacture of a medicament of axotrophin or apolypeptide or polynucleotide encoded by or derived from axotrophin or asubstance that alters amount or activity thereof in an individual, toboost or reduce an aggressive immune response against an antigen or toalter tolerance of the immune system to an antigen.
 5. Use of axotrophinor a polypeptide or polynucleotide encoded by or derived from axotrophinfor assaying immune status.
 6. A method of manipulating the response ofthe immune system to a given antigen in an individual the methodcomprising administering to the individual axotrophin or a polypeptideor polynucleotide encoded by or derived from axotrophin or a substancethat enhances the amount or activity of polypeptide expressed directlyor indirectly by axotrophin.
 6. A method according to claim 5 comprisingpotentiating or increasing the aggressive response of the immune systemof an individual against an antigen, the method comprising administeringto the individual a substance that decreases the amount or activity of apolypeptide expressed directly or indirectly by axotrophin.
 7. A methodfor determining immune status of an individual, the method comprisingdetermining the level of expression of axotrophin or a polypeptide orpolynucleotide encoded by or derived from axotrophin in a test samplecomprising tissue, cells and/or bodily fluid removed or obtained fromthe individual and comparing the level for the test sample with that ofa control sample, wherein a level in the test sample greater than thatof the control sample is indicative that the immune status in theindividual comprises a tolerant immune response, the immune status inthe individual comprises an aggressive immune response.
 8. Isolatedaxotrophin, a polynucleotide or a polypeptide encoded by or derived fromaxotrophin.
 9. A method for producing axotrophin polypeptide comprisinggrowing a culture of cells in a culture medium under conditionspermitting expression of the axotrophin polypeptide, and purifying thepolypeptide from the culture or from the host cells.
 10. A vector forthe expression of axotrophin, a polynucleotide sequence or a polypeptideencoded by or derived from axotrophin, the vector containing axotrophinor a polynucleotide sequence encoding axotrophin, a promoter sequenceand a termination sequence.
 11. A composition containing axotrophin or apolypeptide or polynucleotide encoded by or derived from axotrophin anda pharmaceutically acceptable diluent, carrier or excipient.
 12. Amethod of screening chemical compounds comprising contacting a testsample containing one or more chemical compounds to be screened with abinder selected from axotrophin, a polynucleotide or polypeptide encodedby or derived from axotrophin and fragment of such polynucleotide orpolypeptide and determining whether the chemical compound has bound tothe binder.
 13. A method according to claim 12 for identifying an agentwhich binds to axotrophin or a polypeptide or polynucleotide encoded byor derived from axotrophin comprising: (a) contacting an agent withaxotrophin or a or polynucleotide polypeptide encoded by or derived fromaxotrophin; (b) determining whether the agent binds to the saidpolynucleotide or polypeptide; and (c) detecting the formation of acomplex, formed between the agent and the said polynucleotide orpolypeptide such that if a complex is formed, the agent is detected. 14.A method according to claim 13 in which the compound is contacted withaxotrophin or polypeptide or polynucleotide of axotrophin in a cellwherein the complex drives expression of a receptor gene sequence in thecell, and detecting the complex by detecting reporter gene sequenceexpression.
 15. A kit comprising: i) a polynucleotide or polypeptideprobe and/or monoclonal antibodies said probe or antibodies comprisingaxotrophin or a polypeptide or polynucleotide encoded by or derived fromaxotrophin, and optionally ii) quantitative standards for carrying outmethod according to any one of the preceding claims.
 16. A diagnosticmethod for detecting axotrophin or a polynucleotide or polypeptideencoded by or derived from axotrophin comprising: (a) contacting asample to be tested for the presence of a polynucleotide or polypeptideencoded by or derived from axotrophin with a compound that binds to apolynucleotide or polypeptide encoded by or derived from axotrophin; (b)determining whether the compound binds to a component of the sample; and(c) detecting the formation of a complex, formed between the agent andthe protein or polynucleotide and such that if a complex is formed, thepolypeptide or polynucleotide is detected.
 17. A diagnostic method forassessing the immune response of an individual comprising obtaining atest sample from the individual, incubating the test sample with one ormore of the antibodies to or polynucleotide probes for axotrophin or apolynucleotide or polypeptide encoded or derived from axotrophin andassaying for binding of the polynucleotide probes or antibodies to acomponent within the test sample.
 18. A method according to claim 16 orclaim 17 wherein step (c) comprises testing with an ex vivo immune cellpopulation.
 19. A method according to claim 16 or claim 17 wherein step(c) comprises testing in vivo.
 20. Use of a compound that binds to apolynucleotide or polypeptide encoded by or derived from axotrophin inthe manufacture of a medicament for treating an individual and/or exvivo cell population to modulate immune response to an antigen.